Campus Units

Biochemistry, Biophysics and Molecular Biology, Roy J. Carver Department of

Document Type

Article

Publication Version

Published Version

Publication Date

2016

Journal or Book Title

Journal of Biological Methods

Volume

3

Issue

4

First Page

e53

DOI

10.14440/jbm.2016.144

Abstract

The development of biorenewable chemicals will support green chemistry initiatives and supplement the catalog of starting materials available to the chemical industry. Bacterial fatty acid biosynthesis is being pursued as a source of protein catalysts to synthesize novel reduced carbon molecules in fermentation systems. The availability of methods to measure enzyme catalysis for native and engineered enzymes from this pathway remains a bottleneck because a simple quantitative enzyme assay for numerous enzymes does not exist. Here we present two variations of a fluorescence assay that is readily extendable to high-throughput screening and is appropriate for thiol consuming and generating enzymes including the Escherichia coli enzymes malonyl-coenzyme A transacylase (FabD) and keto-acylsynthase III (FabH). We measured KM values of 60 ± 20 µM (acetyl-CoA) and 20 ± 10 µM (malonyl-ACP) and a kcatof 7.4–9.0 s-1 with FabH. Assays of FabD included a precipitation step to remove the thiol-containing substrate holo-ACP from the reaction product coenzyme-A to estimate reaction rates. Analysis of initial velocity measurements revealed KM values of 60 ± 20 µM (malonyl-CoA) and 40 ± 10 µM (holo-ACP) and a kcat of 2100–2600 s-1for the FabD enzyme. Our data show similar results when compared to existing radioactive and continuous coupled assays in terms of sensitivity while providing the benefit of simplicity, scalability and repeatability. Fluorescence detection also eliminates the need for radioactive substrates traditionally used to assay these enzymes.

Comments

This article is published as Barb, Adam W., and Aaron M. Marcella. "A rapid fluorometric assay for the S-malonyltransacylase FabD and other sulfhydryl utilizing enzymes." Journal of Biological Methods 3, no. 4 (2016): e53. doi: 10.14440/jbm.2016.144.

Creative Commons License

Creative Commons Attribution 3.0 License
This work is licensed under a Creative Commons Attribution 3.0 License.

Copyright Owner

The Authors

Language

en

File Format

application/pdf

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